Materials and methods for inhibiting fouling of surfaces exposed to aquatic environments

ABSTRACT

The subject invention provides materials and methods for inhibiting the biofouling of surfaces exposed to aquatic environments. In one embodiment, the subject invention provides additives for marine paints and surface treatments. The subject invention further provides repellents and selective inhibitors for aquatic and/or terrestrial crustacean pests.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a divisional of application U.S. Ser. No.10/783,312, filed Feb. 19, 2004, now U.S. Pat. No. 7,087,106 whichclaims the benefit of U.S. provisional patent application Ser. No.60/449,098, filed Feb. 20, 2003.

GOVERNMENT SUPPORT

The subject matter of this application has been supported in part byU.S. Government Support under the National Oceanic AtmosphericAssociation Grant No. NA16RG2195. Accordingly, the U.S. Government hascertain rights in this invention.

BACKGROUND OF INVENTION

A variety of organisms settle and colonize surfaces exposed to aquaticenvironments. These include bacteria, algae and sedentary invertebratessuch as bryozoans, sponges, mollusks and barnacles. For example,barnacle larvae are major biofouling animals on marine surfaces.Encrusted barnacle populations increase the fuel requirements for ships,slow their passage and cause deterioration of the painted surfaceleading to corrosion.

Until now, efforts to inhibit the settlement of aquatic organisms havefocused primarily on the inclusion of organotin paint additives that aretoxic to a wide variety of aquatic organisms. With this strategy, theexposed surface must be scrapped and repainted at frequent intervals.Also, although organotin additives to marine paints are effectiveanti-fouling agents, unfortunately they also damage the marineenvironment, killing and sterilizing many free-living organisms wherevessels are docked. The consequent pollution of harbors and coastalmarine waters led to a ban on the use of such additives starting in2003. Safer new antifouling additives are urgently needed to replacethese toxic additives. Thus, there is much interest in finding newmaterials and methods to inhibit the colonization of surfaces by marineand other aquatic organisms.

It would be highly desirable to have anti-fouling additives that aremore selective and more easily degraded such that they are less toxic.As the field evolves towards more selective and less toxic additives,one approach might be to exploit mechanisms associated with the processof settlement rather than with a broad spectrum biocide.

Little is known about the chemoreceptive capabilities of barnaclelarvae, although knowledge of the chemoreceptors of other crustaceans ismore advanced (Strausfield, N., Hildebrand, J. [1999] Curr. Rev.Neurobiol. 9:634-639). Besides being receptive to amino acids, somedecapod crustacean neuronal chemoreceptors are sensitive to certainpyridine compounds, especially 3-substituted pyridines (Hatt, H.,Schmiedel-Jakob I. [1984] J. Comp. Physiol. 154A:855-863; Hatt, H.,Schmiedel-Jakob I. [1985] Chem. Senses 10:317-323; Schmiedel-Jacob I.,Breuninger V., and Hatt H. [1988] Chem. Senses 13:619-632). Some of themost potent 3-pyridyls are natural toxins found in certain nemertines, aphylum of nearly 1,000 recorded species of carnivorous flatworms(Gibson, R. Nemerteans. London: Hutchinson University Library, 1972).Bacq (Bacq, Z. (1936) Bull. Acad. R. Belg. Cl. Sci. (Ser 5)22:1072-1079) first demonstrated that nemertines possess toxins. Severaldecades later the alkaloid anabaseine (FIG. 1A) was isolated from ahoplonemertine (Kem W., Abbott, B., Coates, R. (1971) Toxicon. 9:15-22).Nemertines belonging to this taxonomic class were subsequently found tocontain a variety of pyridyl alkaloids besides anabaseine (Kem, W.(1971) Toxicon. 9:23-32; Kem, W., Scott K., and Duncan J. (1976) Exper.32:684-686; Kem, W. (1988) Hydrobiolog. 156:145-147; Kem, W. (2002) In:Handbook of Neurotoxicology, E. J. Massaro, Ed. Vol. 1. Humana Press,Totowa, N.J., pp.161-193). 2,3′-bipyridyl (BP) was identified as themajor toxic constituent of the chevron nemertine Amphiporus angulatus, acircumboreal species found along northern Atlantic and Pacificcoastlines (Kem, W., Scott K., and Duncan J. (1976), supra; Kem, W.,Soti, F. (2001) Hydrobiolog 456:221-231). The worm uses its armedproboscis to mechanically capture and chemically paralyze its arthropodprey.

2,3′-BP (FIG. 1B) is the only bipyridyl that has been found in livingorganisms, namely tobacco plants and A. angulatus. However, 2,2′-BP,because of its ability to chelate certain heavy metals, is the mostwidely known BP. It is an important industrial product. The insecticidalactivity of 2,3′-BP was noticed many decades ago, but apparently it wasnever marketed as an insecticide (Smith, C., Richardson, D., Shepard H.(1930) J. Econ. Entomol. 23:863-867). Some of the methyl-bipyridyls havebeen found in tobacco leaves and/or tobacco smoke. The5-methyl-2,3′-bipyridyl was found in cured Nicotiana tabacum leaves(Warfield, 1972; Matsushima, 1983) as well as in cigarette smoke. The 6-and the 2′-(or 3-) methyl-2,3′-bipyridyls were also found in cigarettesmoke (Schumacher et al., 1977; Sakuma et al., 1984; Heckman and Best,1981).

Five methyl-2,3′-bipyridyls, including the 4- and5-methyl-2,3′-bipyridyls, have been synthesized by the condensation ofpyridine-3-diazonium chloride with either 4-methylpyridyl or5-methylpyridyl (Frank and Crawford, 1959; Warfield et al., 1972), or bypalladium catalyzed cross-coupling reactions (Ishikura et al., 1984;Bloom, 1990; Jacob et al., 1993). The 6-methyl-2,3′-bipyridyl has alsobeen prepared by the latter methods. The 3-, 5- and2′-methyl-2,3′-bipyridyls have been prepared by catalyticdehydrogenation in gas phase (Bowden, 1969). However, until the practiceof the subject invention, the synthesis of 4′-, 5′, and6′-methyl-2,3′-bipyridyls had not been reported.

BRIEF SUMMARY

The subject invention provides materials and methods for reducing thefouling of surfaces exposed to aquatic environments. In a preferredembodiment, the practice of the subject invention reduces fouling byinhibiting the settlement of aquatic organisms. Advantageously, this canbe achieved without the use of highly toxic compounds. Instead,compounds that deter attachment, but do not necessarily kill theorganisms, are used.

In a preferred embodiment of the subject invention, pyridyl alkaloidsare used to inhibit the settlement (attachment) of aquatic organisms tosurfaces exposed to aquatic environments. The aquatic organisms whosesettlement is inhibited according to the subject invention include, forexample, barnacle larvae.

The compounds useful according to the subject invention includebipyridyls (2,3′-bipyridyls, 2,2′-bipyridyls, anabaseine and variousanabaseine derivatives including, 3-benzylidene-anabaseine, and3-cinnamylidene-anabaseine), tobacco alkaloids (including S- andR-nicotine, myosmine, S- and R-anabaseine and the tripyridylnicotelline) and tetrapyridyls (nemertelline).

A further embodiment of the subject invention pertains to thedevelopment of economical syntheses of anabaseines and bipyridyls.Because of these unique synthesis procedures, the subject inventionprovides relatively inexpensive compounds. These compounds may be used,for example, as additives to marine paints. Advantageously, thesecompounds inhibit settlement without causing a generalized toxic effecton the aquatic environment.

In a specific embodiment of the subject invention, twobipyridyls-2,3′-BP and 2,2′-BP—have been found to be potent inhibitorsof barnacle larval settlement.

Advantageously, the relatively low vertebrate toxicity of the bipyridylsand tetrapyridyl used according to the subject invention, make thesepyridyl alkaloids and related analogs economical and effectiveantifouling additives for the protection of marine surfaces and othersurfaces exposed to aquatic environments.

The pyridyl alkaloids of the subject invention have been found topotently inhibit the settlement of barnacle larvae. While some of thesealkaloids are selectively toxic to crustaceans including barnaclelarvae, others inhibit settlement in a manner that is not directlyrelated to their crustacean toxicity.

Thus, in one embodiment, the subject invention provides additives formarine paints and other such coatings to be applied to surfaces exposedto aquatic environments. The subject invention further providesrepellents and selective inhibitors for aquatic and/or terrestrialcrustacean pests. In this embodiment, the compounds of the subjectinvention can be formulated in any appropriate manner for the control ofthe target pests.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the chemical structure of anabaseine.

FIG. 1B shows the chemical structure of 2,3′-bipyridyl.

FIG. 1C shows the chemical structure of nemertelline.

FIG. 2 shows histogram of barnacle (B. amphitrite) cyprid settlementinhibition by anabaseine, a minor alkaloid of A. angulatus and the majoralkaloid of the tobacco plant Nicotiana glauca (the source of the(S)-anabaseine used in Example 1). Settlement was measured after 24 hrexposure to anabaseine. The IC₅₀ for anabaseine inhibition of settlementin Table 1 was calculated from a probit analysis of this data.

DETAILED DISCLOSURE

The subject invention provides materials and methods for inhibiting thebiofouling of surfaces exposed to aquatic environments. In oneembodiment, the subject invention provides additives for marine paintsand surface treatments. The subject invention further providesrepellents and selective inhibitors for aquatic and/or terrestrialcrustacean pests.

In a preferred embodiment, the practice of the subject inventionprevents attachment of aquatic organisms, such as barnacle larvae, tosurfaces exposed to aquatic environments. Advantageously, the inhibitionof attachment can be achieved with compounds having decreased toxicityto aquatic environments as compared to current methods. Advantageously,the subject invention provides compounds that selectively inhibitsettling of aquatic organisms.

In one embodiment the subject invention provides non-toxic additives forboat paints. Advantageously, the materials of the subject invention canbe used to replace organotin additives that are toxic and whoseapplication has been banned worldwide beginning in 2003. Furthermore,the materials of the subject invention are not expensive to make, arestable, are highly potent and selectively toxic to pests of commercialsignificance.

In a specific embodiment, the subject invention provides compositionsfor application to aquatic surfaces. Compounds of the subject can beformulated with surface treatments and applied directly to any surfaceexposed to an aquatic environment. Examples of surface treatmentsinclude, but are not limited to, paints, varnishes, stains, glosses,glazes, sealants, coatings, and coverings.

In a preferred embodiment of the subject invention, pyridyl alkaloidsand related analogs are used as anti-settlement compounds to inhibit theattachment of aquatic organisms to aquatic surfaces. The compoundsuseful according to the subject invention include bipyridyls(2,3′bipyridyls, 2,2′bipyridyls, anabaseine and various anabaseinederivatives including 3-benzylidene-anabaseines and3-cinnamylidene-anabaseine, tobacco alkaloids (including S- andR-nicotine, myosmine, S- and R-anabaseine and the tripyridylnicotelline), tetrapyridyls (nemertelline), and related analogs andentantiomers. Analogs of 2,3′-BP include methyl substituted 2,3′-BP, forexample, 4′-Me 2, 3′-BP, 5′-Me 2,3′-BP, 6′-Me 2,3′-BP, 3-Me 2,3′-BP, 4Me2,3′-BP, 5-Me 2,3′-BP, and 6Me 2,3′-BP.

Other compounds useful according to the subject invention include thosedescribed in U.S. Pat. No. 5,977,144, which is incorporated herein inits entirety by reference. Advantageously, the relatively low vertebratetoxicity of the bipyridyls, tripyridyls, and tetrapyridyl used,according to the subject invention, make these pyridyl alkaloids andrelated analogs economical and effective antifouling additives for theprotection of marine surfaces and other surfaces exposed to aquaticenvironments. Surfaces that can benefit from the invention include, butare not limited to, boats, piers, docks, buoys, locks, water intakepipes, drainage pipes, fish cages, and jettys.

The pyridyl alkaloids of the subject invention have been found topotently inhibit the settlement of barnacle larvae. While some of thesealkaloids are selectively toxic to crustaceans including barnaclelarvae, others inhibit settlement in a manner that is not directlyrelated to their crustacean toxicity. Thus, these compounds areexcellent antifouling agents that may be used as additives to shippaints and aquatic surface treatments, for example, varnishes, stains,glosses, glazes, sealants, coatings, and coverings.

In a specific embodiment of the subject invention, twobipyridyls-2,3′-BP and 2,2′-BP—have been found to be potent inhibitorsof barnacle larval settlement. Since both are similarly lethal tonauplii after exposure for 24 hours, it is possible that they act upon acommon receptor. However, these two compounds do not necessarily havethe same mechanism of action. In this regard, it is noted that one is aneffective metal chelator and the other is not. Specifically, for2,3′-BP, there is a structure-activity relationship for inhibition thatis not identical with that for acute crustacean toxicity or forinteraction with vertebrate nicotinic receptors.

Anabaseine is a potent nicotinic agonist at most nicotinic receptors andit is able to produce a nicotine-like convulsive paralysis in insectsand crustaceans. 2,3′-BP also can stimulate nicotinic receptors, but atrelatively large concentrations. Because of the low basicity of 2,3′-BP(highest pKa is 4.4), only about one in one thousand molecules isionized at pH 7.4, a common internal pH for vertebrate blood, and evenfewer molecules are ionized at the pH of sea water, which is generallyabout 8.

At physiological pH, anabaseine exists in three different forms: aneutral cyclic imine form chemically similar to 2,3′-BP, a cycliciminium protonated form, and an open-chain (amino-ketone) protonatedform, in roughly equal concentrations (Zoltewicz, J., Bloom L., Kem, W.(1989) J. Org. Chem. 54:4462-4468; Zoltewicz, J., Bloom L., Kem, W.(1990) J. Bioorg. Chem. 18:395-412). On vertebrate nicotinic receptors,the cyclic iminium form was found to be the only one with significantactivity (25; Kem, W. (2002), supra). In a preferred embodiment of thesubject invention, it has been found that, for the nemertine alkaloidsand their isomers, the un-ionized pyridyls are the most effectiveinhibitors of settlement. If the receptors are internal, then thepostulated lack of ionization may enhance penetration to the internalreceptor from the outside. If an inhibitor is to be of practical valueas a paint additive, it would have to be able to enter the cyprid larvaefrom the outside environment.

The comparative potencies of various analogs summarized in Tables 1 and2 suggest that there are several structural requirements for thisactivity. The presence of two heteroatoms in connecting rings isimportant for high potency, since phenylpyridine was essentiallyinactive. Clearly, a cisoid conformation is preferred, since1,9-phenanthroline was as active as 2,3′-BP whereas the transoid1,7-phenanthroline was not nearly as active.

TABLE 1 Larval settlement inhibition and lethal potencies of nemertinepyridyl alkaloids and related analogs Crayfish Lethal Barnacle LarvaeAcute Settlement Median Paralytic Inhibition Conc. Dose Compound IC₅₀(μM) LC₅₀ (μM) PD₅₀ (μg) Nemertine Alkaloids: 2,3′-BP 4.1 (3.2-5.3) 1.9(1.0-4.3) 0.88¹ (0.71-1.1) Anabaseine 1.2 (0.91-1.7)    2 3.6¹ (3.1-4.1)Nemertelline 3.2 (1.8-6.0) — >120 Anabasine 3.0 (1.5-4.9) — 3.9(3.4-4.5) Bipyridyls: 2,3′-BP    4.1    1.9 1.8² (1.6-2.1) 2,2′-BP    7³4.4 (2.9-6.2) >100 2,4′-BP >100  >50 >20 3,3′-BP >100    10 17 (15-19)3,4′-BP >100  >20 61 (53-69) 4,4′-BP >100  >50 11 (9.0-14) AlkaloidAnalogs: 1,9-phenanthroline    2  >50 3.0² (2.2-4.1) 1,7-phenanthroline >50  >50 >30² 2-(3-pyridyl)-  >50  >50 2.6² (2.1-3.2) pyrimidine3-Chloro-2,3′-BP >100  >50 — 2-(Thienyl)-pyridine >100 >100 650²(540-770) 2-(Phenyl)-pyridine >100 >100 520² (400-690) PTHP >100 >10081² (62-107) 1-Methyl-2,3′BP >100 >100 >800² ¹Data published previouslyusing P. clarkii from California (Kem, W., Scott K., and Duncan J.(1976) Exper. 32: 684-686) ²Data published previously using P. clarkiifrom Louisiana (Kem, W., Soti, F. (2001) Hydrobiolog 456: 221-231) ³Theinhibition of settlement data for this compound could not be fit by theprobit method.

TABLE 2 Settlement inhibition and lethal potencies of the eight possibleC-methyl 2,3′-bipyridyls. Inhibition Barnacle Larvae Pot/ SettlementCrayfish Paralysis Paralytic Compound % Inhib Rel. Pot PD₅₀ (μg) Rel.Pot Pot 2,3′-BP 54 (11) 100 1.8 (1.6-2.1) 100 1.0 2′-Me2,3′-BP  0 (33) 0210¹ (160-260) 1 <0.05 4′-Me2,3′-BP 90 (10) 167 69¹ (47-102) 3 565′-Me2,3′-BP 54 (28) 100 3.3 (2.6-4.2) 55 1.8 6′-Me2,3′-BP 30 (16) 56 12(10-15) 15 3.7 3-Me2,3′-BP 34 (13) 63 59¹ (45-78) 30 2.1 4-Me2,3′-BP 83(4) 154 3.6 (2.9-4.4) 50 3.1 5-Me2,3′-BP 95 (5) 176 0.98 (0.7-1.2) 1840.96 6-Me2,3′-BP 16 (16) 30 1.6 (1.2-2.2) 112 0.27 ¹Data publishedpreviously (Kem, W., Soti, F. (2001) Hydrobiolog 456: 221-231)

Using methyl-substituted anabaseines, a structure-activity relationshiphas been delineated for several vertebrate nicotinic receptorsconsistent with the basic Beers-Reich model (Beers, W., Reich, E. (1970)Nature 228:917-922) for interaction with nicotinic cholinergicreceptors. It has been found that a distance of approximately 5Angstroms between an ionizable group and an H-bond electron donor ischaracteristic of nicotinic ligands displaying high affinity for thevertebrate muscle receptor.

2,3′-BP, like anabaseine, may stimulate nicotinic cholinergic receptorsin the crustacean central nervous system. If this is so, then one wouldexpect that methylation of 2,3′-BP would have an effect similar to thatof methylation of anabaseine on vertebrate as well as crayfishreceptors. While methylation can alter N atom basicity as well asintroduce a bulky substituent, the steric hindrance and increasedlipophilicity effects of methylation are expected to dominate. TheC-methylations would not greatly affect the degree of ionization of2,3′-BP at physiological pH.

In the methyl-anabaseine series, the site of methylation has been shownto greatly affect binding to vertebrate nicotinic receptors. Methylsubstituents at the 2′ and 4′ positions of the 3-pyridyl ring greatlyreduce nicotinic receptor activity, probably by preventing the optimalcoplanar relationship of the two rings in anabaseine and 2,3′-bipyridyl.Positioning the methyl at the 3 position of the 2-pyridyl ring alsoreduces activity, but less than the previously mentioned substitutions.While the methyl substitution dependence of crayfish paralytic activity(Table 2) was very similar to that for vertebrate nicotinic receptors,the enhancing effect of methylation at the 4′ position upon barnaclelarval settlement was unique. The high anti-settlement activity of thiscompound, and also of 1,9-phenanthroline and nemertelline (FIG. 1C),suggests that there is some action of the 2,3-BPs that is unrelated toan effect upon nicotinic receptors. One possible non-cholinergic sitewould be the pyridyl chemoreceptors documented to occur in some othercrustaceans.

A variety of nemertine pyridyl alkaloids were tested for inhibition ofbarnacle (Balanus amphitrite) larval settlement and for crustaceans.Eight C-methylated 2,3′-BP isomers were prepared to determine wheresubstitution is permitted without loss of activity. It was determinedthat anti-settlement and toxicity activities were not always related.For instance, 4′-methyl-2,3′-BP displayed only 3% of the crayfishparalytic activity of 2,3′-BP but inhibited settlement almost 2-foldmore effectively. Two other isomers displaying exceptionalanti-settlement activity were the 4- and 5-methyl-2,3′-BPs; these alsodisplayed high crustacean toxicity. Nemertelline inhibited barnaclesettlement at concentrations similar to 2,3′-BP but was 136-fold lesstoxic when injected into crayfish. Nicotelline, a tripyridyl, displayedan activity similar to nemertelline. Thus, certain bipyridyls andtetrapyridyis can be used according to the subject invention asanti-fouling additives.

A further embodiment of the subject invention pertains to thedevelopment of economical syntheses of anabaseines and bipyridyls. Oneof the most attractive aspects of these compounds as anti-settlementadditives is their structural simplicity and ease of chemical synthesis.Many substituted bipyridyls have already been synthesized andinvestigated. However, syntheses of all eight possible C-methyl isomersof 2,3′-bipyridyl have not been known until the subject invention. In aspecific embodiment of the current invention, a method to synthesize the4′-, 5′- and 6′-methyl-2,3′-bipyridyls is disclosed. Advantageously, theprecursor materials, methyl-anabaseines, for the formation of the 4′,5′-and 6′-methyl-2,3′-bipyridyls are commercially available. Because ofthese unique synthesis procedures, the subject invention providesrelatively inexpensive compounds, which can be utilized as additives toaquatic surface treatments and paints.

Although other natural products that are potent inhibitors of settlementare known, they are not as attractive as commercial products. They areless attractive because they have more complicated structures; thismakes them much more expensive to produce. Also, their environmentalfates and effects are more difficult to determine.

A further embodiment of the subject invention is directed to methods ofinitiating paralysis in humans and animals. This specific embodiment isbeneficial in clinical settings where a patient is required to be stillwhile undergoing medical treatments. One method of initiating paralysiscomprises injecting a patient with a sufficient amount of a compositionof a pharmaceutically acceptable carrier and a pyridyl alkaloid, apharmaceutically acceptable salt or analog thereof. A pyridyl alkaloidis selected from the group consisting of 2,3′-bipyridyl, 2,2′-bipyridyl,anabaseine, 3-benzylidene-anabaseine, 3-cinnamylidene-anabaseine,S-nicotine, R-nicotine, myosmine, S-anabasine, R-anabasine, nicotelline,nemertelline, 1-9-phenanthroline, 4′-Me2,3′-bipyridyl,5′-Me2,3′-bipyridyl, 6′-Me2,3′bipyridyl, 3-Me2,3′-bipyridyl,4-Me2,3′-bipyridyl, 5-Me2,3′-bipyridyl, 6-Me2,3′-bipyridyl,2,4′-bipyridyl, 3,3′-bipyridyl, 3,4′-bipyidyl, 4,4′-bipyridyl,2-(3-pyridyl)-pyrimidine, 2′-Me2,3′-bipyridyl, and salts, analogs,enantiomers and mixtures thereof.

MATERIALS AND METHODS Compounds

2,3′-Bipyridyl (2,3′-BP), 2-(thienyl)-pyridine and 2-(phenyl)-pyridinewere purchased from Aldrich Chemical Company. TheC-methyl-2,3′-bipyridyls were prepared from the correspondingmethylanabaseines by dehydrogenation. Description of the synthesis of1-methyl-2,3′-BP has been reported (Zoltewicz, J., Bloom L., Kem, W.(1992) J. Org. Chem. 57:2392-2395). Compounds were purified by silicagel chromatography as their free bases, and were homogeneous asevaluated by silica gel TLC, reversed phase HPLC and NMR analysismethods.

Biological Assays

Adult freshwater crayfish Procambarus clarkii obtained from Louisianawere used as previously described (Kem W., Abbott, B., Coates, R.(1971), supra; Kem, W. (1971), supra; Kem, W., Scott K., and Duncan J.(1976), supra). Each alkaloid, dissolved in 0.9% sodium chloridecontaining 10 mM Tris buffer (pH 7.4), was injected into the haemocoelewhich surrounds the heart. Dose per unit weight was kept constant byinjecting a variable volume, 0.10 ml per 10 g fresh weight. Paralysiswas measured 15 minutes after injection by measuring the animal'sability, after being placed on its back, to right itself within a2-minute period. Although the convulsive paralytic effect of thebipyridyls reached a peak about 2-minutes after injection, paralysiscould be more conveniently measured 15 minutes after injection.Generally, the same number (10-20) of crayfish were injected at eachdose, and the dose interval was kept constant. Before the assay,crayfish were tested to ascertain that they could right themselveswithin a one minute period. The median paralytic dose (PD₅₀) wascalculated using the Spearman-Karber statistical method (Kem W., Abbott,B., Coates, R. (1971), supra).

The barnacle larval 24 hour settlement and toxicity assays were carriedout using previously described protocols (Rittschof, D., Clare, A.,Gerhart, D., Mary S., Bonaventura, J. (1992) Biofoul 6:115-122;Sasikumar, N., Clare, A., Gerhart, D., Stover, D., Rittschof, D. (1995)Bull. Environ. Contam. Toxicol. 54:289-296). While the cypris larvalstage was used in settlement tests, the second naupliar larval stage wasused in toxicity tests. Great care was taken to ensure that the freebases completely dissolved in aged sea water solutions previouslyfiltered to remove particles >than 100 kDa. This was accomplished bydiluting (by immediate and thorough vortexing) a 3×10⁻² Molar solutionof the free base in methanol at least 1,000-fold into the filtered seawater. Diluted aqueous solutions of the compounds were not refrigeratedand were used within an hour of preparation.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures, to the extent they are notinconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Inhibition of Barnacle Cyprid Settlement

The initial assays with the nemertine alkaloids showed that these3-substituted pyridly alkaloids were able to inhibit settling atmicromolar concentrations (Table 1). These included anabaseine and2,3′-BP. Nemertelline also inhibited larval settlement. Thistetrapyridyl is essentially composed of two 2,3′-BP moieties.(S)-anabaseine (FIGS. 1, 2) also inhibited settlement.

All six possible unsubstituted bipyridyls were also tested. The assays(Table 1) showed that 2,3′-BP potently inhibited barnacle settlement andwas also the most toxic as evaluated by barnacle naupliar 24 hrmortality and 15 minute crayfish paralysis. 2,2′-BP inhibited settlementat only slightly higher concentrations, and its 24 hour naupliartoxicity was also slightly less, but it displayed a remarkably lowtoxicity on crayfish as measured by the paralysis assays. The other fourBPs displayed very weak ability to inhibit larval settlement, relativeto these two isomers.

Various analogs of 2,3′-BP were then tested to determine what molecularfeatures are important for anti-settlement activity. Amongst thesecompounds 1,9-phenanthroline, a rigid analog containing the “cisoid”conformation of 2,3′-BP, was almost as active as 2,3′-BP. In contrast,1,7-phenanthroline, a rigid analog containing the “transoid”conformation of 2,3′-BP, was inactive at a 10-fold higher concentration.These differences indicate that the most biologically effectiveconformation of 2,3-BP is cisoid, where the two nitrogen atoms areoriented on the same side of the planar molecule.

The inactivity of 2-(phenyl)-pyridine indicates that both nitrogen atomsof 2,3′-BP are important for this activity. The inactivity of the2-(thienyl)-pyridine indicates that a sulphur heteroatom will noteffectively replace the one nitrogen in 2,3′-BP. The inability of the3-chloro-2,3′-BP to inhibit settlement at 100 μM contrasts with therelatively small loss of activity when a methyl group is attached atthis position (Table 2). Since the chlorine atom is not too different insize from the methyl group, it seems that the electron-withdrawingproperty of the chlorine is detrimental to activity; the methyl group incontrast would be electron-donating and would increase the basicity ofthe nitrogen on the 2-pyridyl ring of 2,3′BP.

PTHP (full name: 2-(3-pyridyl)-3,4,5,6-tetrahydropyrimidine) is apermanently charged analog of anabaseine and 2,3′-BP; its relativeinactivity indicates that protonation of the 2-pyridyl ring nitrogeninhibits activity. The low anti-settlement activity of anotherpermanently ionized analog, 1-methyl-2,3′-BP, is also consistent withthis interpretation.

The relative abilities of the eight possible C-methylated 2,3′-BPs toinhibit settlement were determined at a single concentration in threeseparate experiments, as shown in Table 2. What was most remarkable wasthe enhanced anti-settlement potency of the 4′-Me-BP and, as a result ofthe same substitution, a greatly reduced (33-fold) acute crayfishtoxicity. Placement of a methyl at either the 4 or 5-positions of the2-pyridyl ring also elevated anti-settlement activity. Additionally, oneobserves that placement of a methyl group at either of the ortho-(2′ or6′) positions of the 3-pyridyl ring reduced anti-settlement activity,particularly when a methyl substituent was added at the 2′-position.

EXAMPLE 2 Crayfish Paralytic Activities

Crayfish paralysis is a simple bioassay which allows quantitativecomparison of the acute neurotoxicity of a variety of toxins (Kem W.,Abbott, B., Coates, R. (1971), supra). It has been particularly usefulfor examining nicotinic compounds, since these receptors are mostlylocated within the central nervous system; where electrophysiologicalrecordings of synaptic responses generated by nicotinic receptors aremore difficult (Wiersma, C., Schallek, W. (1946) J. Neurophysiol10:23-38; Prosser, C. (1940) J. Cell Comp. Physiol. 16:25-38). Since thecrustacean nervous system does not seem to possess an equivalent of thevertebrate blood: brain barrier, chemicals injected into the systemiccirculation should readily reach the central nervous system (Abbott, N.(1970) Nature 225:291-293).

Injected anabaseine and 2,3′-BP both caused a remarkably rapidconvulsive paralysis of crayfish and other crustaceans. Animalsinitially develop tremors, then flex their legs, repeatedly flip theirtails, then fall on their side in a tonic convulsion within 2-5 minutesafter injection. Recovery is appreciable within 15 minutes, the timeselected as most convenient for evaluating groups of 5 animals rapidlyinjected one after another. Thus the median paralytic doses reported inthe two tables are actually more than twice the PD₅₀ values which wouldbe obtained at minute post-injection, with the exception ofnemertelline. 2,2′-BP was at least 50-fold less toxic than 2,3′-BP whenmeasured by this bioassay. Nemertelline was about 140-fold lessparalytic than 2,3′-BP. Anabaseine was approximately 4-fold lessparalytic than 2,3′-BP or anabaseine.

The most paralytic alkaloid analogs were 1,9-phenanthroline and2-(3-pyrimidinyl)-pyridine, whose structures are very similar to that of2,3′-BP. These compounds also are un-ionized at physiological pH. Thelow activity of the transoid isomer 1,7-phenanthroline indicates thatparalysis, like settlement inhibition, is affected by the cisoidconformation of 2,3′-BP. Anabaseine was slightly less paralytic than2,3′-BP; this may be due to its occurrence in several forms atphysiological conditions. The permanently ionized analogs of 2,3′-BP(PTHP and 1-methyl-2,3′-BP) displayed very low paralytic activity aswell as anti-settlement activity.

The effects of individually replacing each hydrogen atom in2,3′-bipyridyl with the more bulky methyl substituent was alsoinvestigated. In Table 2 one observes the methylation of carbonsadjacent to the ring-joining carbons (2′,4′ and 3) greatly reducedparalytic activity, as has also been observed for methyl-anabaseinestimulation of nicotinic receptors in vertebrates. On the other hand,methyl substitution at the 4,5 and 6 positions of the 2-pyridyl ring andthe 5′ and 6′ positions of the 3-pyridyl ring did not greatly affectparalytic potency. In one instance (5 position), methylation evenincreased paralytic potency almost 2-fold. Thus, the effects ofmethylating 2,3′-BP on crayfish paralytic activity were in someinstances quite different from what was observed for inhibition ofbarnacle larval settlement.

EXAMPLE 3 General Synthesis of Methyl-2,3′-bipyridyls

A mixture of methyl-anabaseine free base (35 mg, 0.2 mmole) andN-chlorosuccinimide (67 mg, 5 mmole) in carbon tetrachloride (5 ml) wasstirred overnight at room temperature. Water (3 ml) and sodium hydrogencarbonate (0.3 g) were added, separated and the aqueous phase wasextracted with carbon tetrachloride (2×1 ml). The combined organicphases were dried (magnesium sulfate) and evaporated under vacuum givingthe methyl-3,3-dichloro-anabaseines in 68-98% yield. These intermediateswere pure enough by TLC and ¹H-NMR for the next step. The abovemethyl-3,3-dichloro-anabaseines were dissolved in a solution of sodiummethoxide in dry methanol (0.5 ml of a 2M solution, 1.0 mmole) andstirred at room temperature overnight. After evaporation in vacuum tothe residue water (2 ml) was added and extracted with dichloromethane(5×1 ml). The combined organic solutions were dried (magnesium sulfate),decolorized (activated carbon), and evaporated in a vacuum (2 mm Hg, 45°C.) giving the pure methyl-2,3′-bipyridyls as light brown oils in 64-84%yield.

Specifically, the yields for 4′-Methyl-2,3′-bipyridyl,5′-Methyl-2,3′-bipyridyl, and 6′-Methyl-2,3′-bipyridyl are 72%, 64%, and84% respectively in applying the process of the subject invention.

In addition, the NMR data of the dichloro intermediates and themethyl-bipyridyls are presented in Tables 3 and 4, respectively, whilethe GC and MS data of methyl-bipyridyls are presented in Table 5.

TABLE 3 NMR data of 3,3-dichloro-2,3′-bipyridyl and its methylderivatives Position of the H Compound 2′ 4′ 5′ 6′ 4 5 6 CH₃3,3-Dichloro- 9.17, d* 8.37, ddd 7.39, ddd 8.65, dd 2.75-2.81, m2.00-2.10, m 3.98, dd — 2,3′-BP 1.8 8.1, 1.8, 1.5 8.1, 5.1, 0.6 5.1, 1.56.0, 6.0 3,3-Dichloro- — 7.96, dd 7.18, dd* 8.53, dd 2.76-2.82, m2.03-2.12, m 3.94, dd 2.56, s 2′-Me-BP 7.8, 1.8 7.8, 5.1 5.1, 1.8 6.0,6.0 3,3-Dichloro- 8.88, s — 7.46, d 8.57, d 2.78-2.84, m 2.05-2.14, m3.97, dd 2.46, s 4′-Me-BP 5.4 5.4 6.0, 5.7 3,3-Dichloro- 9.00, d* 8.13,s* — 8.48, d* 2.74-2.80, m 2.00-2.10, m 3.97, dd 2.41, s* 5′-Me-BP 1.51.5 6.0, 5.7 3,3-Dichloro- 9.04, d 8.19, dd 7.18, d — 2.74-2.80, m1.99-2.08, m 3.95, dd 2.61, s 6′-Me-BP 2.1 8.1, 2.1 8.1 6.0, 6.03,3-Dichloro- 9.12, d* 8.30, ddd 7.34, ddd 8.62, dd 2.43-2.56, m1.74-1.98, m 4.17, ddd, (1H) 1.39, d 4-Me-BP 1.8 8.1, 1.8, 1.8 8.1, 4.8,0.9 4.8, 1.8 (1H) (2H) 19.5, 5.4, 1.8 6.6 3.79, ddd, (1H) 19.5, 10.8,6.0 3,3-Dichloro- 9.15, d* 8.29, ddd 7.32, ddd 8.64, dd 2.85-2.91, m2.28-2.37, m 4.16-4.26, m 1.07, d 5-Me-BP 2.1 8.1, 2.1, 1.5 8.1, 4.8,0.6 4.8, 1.5 (1H) (1H) (1H) 5.7 2.28-2.37, m 3.24-3.36, m (1H) (1H)First row: chemical shift in ppm and its splitting, *line broadening orfurther unresolved small long range coupling(s); second row: couplingconstant(s) in Hz. In parentheses: the number of hydrogen(s), if it isnecessary.

TABLE 4 NMR data of 2,3-bipyridyl and its methyl derivatives Position ofthe H Compound 2′ 4′ 5′ 6′ 3 4 5 6 CH₃ 2,3′-BP 9.20, dd 8.33, ddd 7.41,ddd 8.66, dd 7.74-7.84, m 7.74-7.84, m 7.30, ddd 8.73, ddd — 2.4, 0.97.8, 2.4, 1.8 7.8, 4.8, 0.9 4.8, 1.8 6.9, 4.8, 1.8 4.8, 1.8, 1.22′-Me-BP — 7.73, dd 7.23, dd* 8.55, dd 7.43, ddd 7.78, ddd 7.30, ddd8.73, ddd 2.61, s 7.8, 1.8 7.8, 4.8 4.8, 1.8 7.8, 1.2, 0.9 7.8, 7.8, 1.87.8, 4.8, 1.2 4.8, 1.8, 0.9 4′-Me-BP 8.59, s* — 7.21, d* 8.49, d 7.43,ddd 7.80, ddd 7.31, ddd 8.73, ddd 2.41, s 4.8 5.1 7.8, 1.2, 0.9 7.8,7.5, 1.8 7.5, 4.8, 1.2 4.8, 1.8, 0.9 5′-Me-BP 8.98, d 8.15, s* — 8.48,d* 7.71-7.81, m 7.71-7.81, m 7.27, ddd 8.71, ddd 2.41, s 2.1 1.8 6.6,4.8, 1.8 4.8, 1.5, 1.2 6′-Me-BP 9.07, d 8.22, dd 7.23-7.29, m —7.69-7.80, m 7.69-7.80, m 7.23-7.29, m 8.70, ddd 2.62, s 2.1 8.1, 2.44.8, 1.8, 1.2 3-Me-BP 8.80, dd 7.89, ddd 7.40, ddd 8.64, dd — 7.62, dd*7.24, dd 8.56, dd* 2.39, s 2.4, 0.6 7.8, 2.4, 1.8 7.8, 4.8, 0.6 4.8, 1.87.8, 0.9 7.8, 4.8 4.8, 0.9 4-Me-BP 9.17, dd 8.31, ddd 7.39, ddd 8.64, dd7.56, s* — 7.12, d* 8.57, dd 2.44, s 2.4, 0.9 7.8, 2.4, 1.5 7.8, 4.8,0.9 4.8, 1.5 5.1 5.1, 0.6 5-Me-BP 9.17, dd 8.28, ddd 7.37, ddd 8.61, dd7.55-7.66, m 7.55-7.66, m — 8.53-8.55, m 2.37, s* 2.4, 0.9 8.1, 2.4, 1.88.1, 4.8, 0.9 4.8, 1.8 6-Me-BP 9.18, dd 8.30, ddd 7.37, ddd 8.62, dd7.52, d* 7.65, dd 7.13, d* — 2.62, s 2.4, 0.9 8.1, 2.4, 1.8 8.1, 4.8,0.9 4.8, 1.8 7.8 7.8, 7.5 7.5 First row: chemical shift in ppm and itssplitting; *Line broadening or further unresolved small long rangecoupling(s); second row coupling constant(s) in Hz.

TABLE 5 GC retention time and MS data of 2,3-bipyridyl and its methylderivatives CH₃ Position 2,3′-BP 2′ 4′ 5′ 6′ 3 4 5 6 (m:s) m/z 12:1612:17 12:43 13:19 12:52 12:43 13:15 13:21 12:46 51 5.3 6.5 7.8 7.7 6.663 5.4 5.3 5.3 6.5 5.1 65 7.1 7.0 8.0 75 5.1 78 8.2 8.1 79 6.4 89 5.45.8 92 7.4 5.5 6.6 104 5.2 115 9.5 15.6 9.7 8.0 11.1 11.4 13.7 11.2 1177.0 7.9 5.8 118 5.3 128 7.7 8.3 130 19.0 141 5.1 6.5 10.1 6.5 5.4 5.6142 12.1 12.3 19.6 15.8 21.3 12.0 13.3 12.9 143 12.7 8.4 5.2 6.9 9.4 14410.1 7.3 32.1 36.9 23.1 155 34.6 40.8 9.2 156 5.3 168 15.8 14.3 6.8 5.611.5 9.1 6.7 5.5 169 100.0 100.0 65.4 61.7 100.0 72.5 77.6 68.8 170 19.619.6 100.0 100.0 15.0 100.0 100.0 100.0 171 16.6 16.2 15.3 15.5 12.7

EXAMPLE 4 Synthesis of 3-Hydroxymethyl-2,3′-bipyridyl

To a continually stirred, ice-cold solution of2,3′-bipyridine-3-carboxylic acid (0.28 g, 1.40 mmole) in drytetrahydrofuran (10 ml) and triethylamine (0.20 ml, 1.43 mmole) ethylchloroformate (0.14 ml, 1.46 mmole) was added drop-by-drop over a 30minute period. The precipitated product was then filtered and washedseveral times with tetrahyrofuran (3×2 ml). The THF phases werecombined, cooled to +10° C., and while stirring under an argonatmosphere, sodium borohydride (0.185 g, 4.9 mmole) was added and thenmethanol (0.90 ml, 22.2 mmole) was added drop-by-drop over a 1 hourperiod during which time the temperature increased to 20° C. (Soai etal., 1987). The material was stirred for an additional 1 hour at roomtemperature. Then, 1N hydrochloric acid was carefully added, the solventwas evaporated under vacuum to 5 ml and washed with dichloromethane (3×5ml). Sodium carbonate (0.85 g) was added to the aqueous phase, which wasthen extracted with dichloromethane. The combined fractions were driedwith magnesium sulfate and evaporated under vacuum (2 Hg mm, 45° C.)giving a pure product (0.22 g, 84%). ¹H-NMR, δ, ppm, 8.71 (1H, dd,J=2.1, 0.6 Hz, C2′-H), 8.55 (1H, dd, J==4.8, 1.5 Hz, C6-H), 8.52 (1H,dd, J=4.8, 1.8 Hz, C6′-H), 7.95 (1H, dd, J=7.8, 1.5 Hz, C4-H), 7.91 (1H,ddd, J=7.8, 2.1, 1.8 Hz, C4′-H), 7.35 (1H, ddd, J=7.8, 4.8, 0.6 Hz,C5′-H), 7.31 (1H, dd, J=7.8, 4.8 Hz, C5-H), 4.58 (2H, s, CH₂-O).

EXAMPLE 5 Synthesis of 3-Methyl-2,3′-bipyridyl

A 3-hydroxymethyl-2,3′-bipyridyl (42.3 mg, 0.227 mmole) solutioncontaining methanol (5 ml) and 4N solution of hydrogen chloride in dry1,4-dioxane (0.57 ml, 2.3 mmole) was hydrogenated in the presence ofpalladium on activated carbon catalyst (10%, 40 mg) at room temperatureand ambient pressure for 3 hours. The catalyst was filtered off, washedwith methanol (3×1 ml), and the combined methanolic solutions wereevaporated under vacuum. The residue was dissolved in water (2 ml),sodium carbonate was added (0.1 g) and the solution was extracted withdichloromethane (3×2 ml). The combined dichloromethane solutions weredried (magnesium sulfate) and rotary evaporated at 2 mm Hg-45° C.,giving the pure product (29.4 mg, 76%) as a colorless oil.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A compound which is 4′-Me2,3′-bipyridyl.